Susceptibility of Genomic Imprinting to ART

Mellissa MannChildren’s Health Research Institute

Department of Obstetrics & Gynecology, and Biochemistry

University of Western Ontario

35-70 million couples involuntarily infertile

1 in 6 people of reproductive age

ART

1-2% children born by ART

Infertility and Assisted Reproductive Technologies

Children conceived via ARTs are at increased risk

Intrauterine growth retardation Premature birth Low birth weight Possibly genetic disorders

ART-induced perturbations in the mouse

Reduced viability Intrauterine growth retardation Developmental abnormalities Deviation in behaviour

Developmental abnormalities in in vitro produced livestock

Large Offspring Syndrome

• Higher perinatal mortality

• Breathing difficulties• Reluctance to suckle• Skeletal anomalies• Large organs• Cerebellar dysplasia

•Increased prenatal loss•Large placentas•Large fetuses•Polyhydramnios•Parturition problems

Duke University Medical Center

Epigenetics: Heritable alterations in gene activity without a change in DNA sequence

Obese and YellowSkinny and Brown

Epigenetics

EstrogenDisruptors

HerbicidesPesticides

Nutrition DrugsVitamins

Epigenetics: Mediator of Environment, Development, and Disease

Pathology

Cancer

CardiovascularDisorders

GrowthDisorders

ImprintingDisorders

Lupus

NeurologicalDisorders

PediatricDisorders

ReproductiveDisorders

Paternal alleleMaternal allele

Genomic ImprintingThe unequal expression of the maternal and paternal alleles of

a gene

Imprinted or markedwith their gametic-origin

Maternal allele Paternal allele

Mouse/Human Imprinted Domains

Kcnq1

Kcnq1ot1

Cd81

Ascl2Th Ins2H19

Igf2Nap1l4

Slc22a1l

Tssc3 Cdkn1c

Mouse Distal 7/ Human 11p15.5

Beckwith-Wiedemann Syndrome Critical Region 1

IC IC

Prader-Willi Syndrome Angelman Syndrome

Frat3

Magel2NdnMouse Central 7/ Human 15q11-13

Ipw Ube3A Atp10c

Mkrn3

SnurfSnrpn snoRNA Genes

IC

IC Antisense

Paternal MaternalBiallelic Imprinting Center

IC Non-coding RNA

Methylated imprinted allele

Unmethylated imprinted allele

PaternalGenome

MaternalGenome

Methylation changes during mouse preimplantation development

Are genomic imprints maintained in preimplantation embryos after in vitro

culture?

CASTChromosome 7

B6(CAST7)

B6

B6

X CAST B6

B6(CAST7) mice for use in allelic analyses

F1

Peg3

Snrpn

H19

Kcnq1ot1

Trophectoderm

ICM

Blastocyst

Whitten’s

KSOMaa

2-cell

In vitro preimplantation culture regimes

PaternalMaternal

14%p=0.002

6%p=0.006

63%

Loss of H19 imprinted expression occurs in a subset of individual

blastocysts after culture in Whitten’s medium

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Vivo

KSOMaa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Whitten’s

10080

6040200

100

80604020

0

100

8060

4020

0

100806040200

100

8060

4020

0

Blastocyst

Loss of imprinting occurred during preimplantation development in culture, indicating that mechanisms that operate to maintain imprinting were disrupted.

What are the long-term effects of preimplantation development in

culture?

Cultured Blastocysts

Recipient Mothers

Postimplantation embryos recovered at 9.5 days of pregnancy after preimplantation culture in

Whitten’s medium.

8%3% 3% 5%

81%

10%

30%

11% 14%

32%

0%

20%

40%

60%

80%

100%

Empty SeverelyAbnormal

Abnormal Delayed Normal

Controls Whitten's

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Embryo Placenta

H19

Ascl2

Snrpn

Peg3

1008060

40

200

100

8060

40

20

0

100

80

6040

200

100

80

60

4020

0

Paternal

Maternal

Whitten’sKSOMaa Whitten’sKSOMaaVivoVivo

Loss of imprinted expression occurs primarily in day 9.5 placentasafter preimplantation culture

Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture

100 10087

100

7592

825

13

0

20

40

60

80

100

100 100 92 10088

55

12

45

8

0

20

40

60

80

100

Embryo Placenta

H19

Ascl2

Snrpn

Peg3

WK WKVV

Paternal

Maternal

Biallelic

100

7592

825

0

20

40

60

80

100

100 10083

100

6373

372716

0

20

40

60

80

100

Perturbations in imprinting persist long after embryos have been removed from culture.

Loss of imprinting occurs more frequently in extraembryonic than embryonic lineages

Placenta

Embryo

Day 9.5

Trophectoderm

ICM

Blastocyst

Loss of impr

inting

Maintains/restores

imprinting

Preimplantation

Culture Proximity to culture?

ICM vs TE?Less redundancy?

Sporadic imprinting defects may arise during ART procedures

Beckwith-Wiedemann Syndrome

X XOR OR

Biparental origin

ART children diagnosed with imprinting disorders

Loss ofmaternalmethylation

Loss of maternalmethylation

Normal

X

IM

OR OR

Angelman Syndrome

X X

Biparental origin

PGC Oocytematuration

HumansSuperovulation

+/- GnRH treatment

Oocyte retrieval

IVF/ICSI

In vitro culture

Superovulation In vitro culture

Mice

Assisted Reproductive Technologies

PGC Oocytematuration

Superovulation In vitro cultureMice

Future StudiesAssisted Reproductive Technologies

1

1. Do imprinting defects result from superovulation?

2,3

2. When is imprinting lost during preimplantation development in culture?

3. Does loss of imprinting occur in mouse embryos cultured in media used in human assisted reproduction?

4,5

4. Is loss of imprinting tissue-specific?

5. What are the long-term affects of preimplantation culture on genomic imprinting and development?

1. Determine whether superovulation contributes to loss of imprinting

Spontaneous vs Induced Ovulation

Imprint Acquisition

Extraovarian Sensitivity

Preantral Early antral Preovulatory

2. Determine when imprint maintenance is lost during preimplantation development in

culture.

Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3

In vitro culture

3. Does loss of imprinting occur in mouse embryos cultured in media used in human

assisted reproduction? Trophectoderm

ICM

Blastocyst

Global

G1.2/G2.22-cell

HTF

P-1/Bl +SSS

One Step

Two Step

H19Snrpn Peg3Kncq1ot1

H19SnrpnPeg3Kncq1ot1

Day 5.5 Day 6.5 Day 7.5 Day 8.5

Right horn

4. Determine how disruptions lead to the selective loss of imprinting in the

placenta

In VivoderivedWhitten’s

Left horn Right horn

Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3

Ultrasound biomicroscopyDay 9.5, Day 13.5, Day 17.5

In VivoderivedWhitten’s

Left horn Right horn

5. Determine the long-term affects of preimplantation culture on genomic

imprinting and development

U

D

E

P

U

D

U

D

EP

UD

NTNT

Ultrasound biomicroscopy at Day 9.5-10.5 of gestation Growth and Viability

Crown-rumplength

Variable embryonic growth Calcium hydroxyapatite deposits

Resorption

Beckwith-Wiedemann Syndrome

Macrosomia

Visceromegaly (liver, heart)

Macroglossia ( tongue)

Abdominal wall defects

Polydactyly (limb)

Cardiac defects (heart)

Adrenal defects

Lens defects (eye)

Vertebral defects

Hemihypertrophy

Polyhydramnios

Placentomegaly/Placental hydrops

Ultrasound biomicroscopy at Day 13.5 of gestation

Limb

Heart Eye

Tongue

Liver

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Michael GoldingAnne PinLiyue ZhangSarah LaloneJulia FosterBrenna MarketLauren Magri

Michelle GabrielMorgan McWilliam

Acknowledgements

University of Pennsylvania

Marisa BartolomeiRichard Schultz